Improved production of alkaline polygalacturonate lyase by homologous overexpression pelA in Bacillus subtilis

A polygalacturonate lyase (PGL), PelA, was purified from the culture broth of Bacillus subtilis 7-3-3, with a molecular weight, optimal temperature, and pH of approximately 45 kDa, 55 °C, and 9.4, respectively. The PGL gene (pelA) was homologously overexpressed in B. subtilis 7-3-3 to increase the gene copies and enhance the PGL production. The resulting PGL activity was 2138 U mL^?1 at 44 h, and the productivity reached 48.58 U (mL h)^?1 through the homologous overexpression of strain B-pN-pelA in a 7.5 L fermentor, the highest PGL production compared to those reported in literature to the best of our knowledge. Crude enzyme has high PGL and PGase activity, which can remove 50.58% of pectin in unpretreatment ramie fibers at 50 °C for 4 h. Meanwhile, the enzyme system with a low level hemicellulase and almost no cellulase will further help in enhancing the efficiency of degumming besides maintaining tenacity of plant fiber. The B. subtilis B-pN-pelA shows high genetic stability and has great potential in the textile industry.     

Improvement of catalytical properties of two invertases highly tolerant to sucrose after expression in Pichia pastoris. Effect of glycosylation on enzyme properties

Zymomonas mobilis genes encoding INVA and INVB were expressed in Pichia pastoris, under the control of the strong AOX1 promoter, and the recombinant enzymes were named INVA_AOX1 and INVB_AOX1. The expression levels of INVA_AOX1 (1660 U/mg) and INVB_AOX1 (1993 U/mg) in P. pastoris were 9- and 7-fold higher than those observed for the native INVA and INVB proteins in Z. mobilis. INVA_AOX1 and INVB_AOX1 displayed a 2- to 3-fold higher substrate affinity, and a 2- to 200-fold higher catalytic efficiency (k_cat/K_M) than that observed for native INVA and INVB from Z. mobilis. Positive Schiff staining of INVA_AOX1 and INVB_AOX1 suggested a glycoprotein nature of both invertases. After deglycosylation of these enzymes, denoted D-INVA_AOX1 and D-INVB_AOX1, they exhibited a 1.3- and 3-fold lower catalytic efficiency (107 and 164 s⁻¹ mM⁻¹, respectively), and a 1.3- to 5-fold lower thermal stability than the glycosylated forms at temperatures of 35–45 °C. After deglycosylation no effect was observed in optimal pH, being of 5.5 for INVA_AOX1, INVB_AOX1, D-INVA_AOX1 and D-INVB_AOX1. The invertase activity of both enzymes increased in 80% (INVA_AOX1) and 20% (INVB_AOX1) in the presence of Mn²⁺ at 1 mM and 5 mM, respectively. INVA_AOX1 and INVB_AOX1 were highly active at sucrose concentrations of up to 400 and 300 mM, respectively; however, the tolerance to sucrose decreased to 300 mM for D-INVA_AOX1. Our findings suggest that glycosylation of INVA_AOX1 and INVB_AOX1 plays an important role in their thermal stability, catalytic efficiency, and tolerance to sucrose. In conclusion, the expression of INVA and INVB from Z. mobilis in P. pastoris yields new catalysts with improved catalytic properties, making them suitable candidates for a number of industrial applications or for the improvement of ethanol production from cane molasses.     

The effect of temperature on protein refolding at high pressure: Enhanced green fluorescent protein as a model

The application of high hydrostatic pressure (HHP) impairs electrostatic and hydrophobic intermolecular interactions, promoting the dissociation of recombinant inclusion bodies (IBs) under mild conditions that favor subsequent protein refolding. We demonstrated that IBs of a mutant version of green fluorescent protein (eGFP F64L/S65T), produced at 37 °C, present native-like secondary and tertiary structures that are progressively lost with an increase in bacterial cultivation temperature. The IBs produced at 37 °C are more efficiently dissociated at 2.4 kbar than those produced at 47 °C, yielding 25 times more soluble, functional eGFP after the lower pressure (0.69 kbar) refolding step. The association of a negative temperature (−9 °C) with HHP enhances the efficiency of solubilization of IBs and of eGFP refolding. The rate of refolding of eGFP as temperature increases from 10 °C to 50 °C is proportional to the temperature, and a higher yield was obtained at 20 °C. High level refolding yield (92%) was obtained by adjusting the temperatures of expression of IBs (37 °C), of their dissociation at HHP (−9 °C) and of eGFP refolding (20 °C). Our data highlight new prospects for the refolding of proteins, a process of fundamental interest in modern biotechnology.     

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

A xylanase produced by Thermomyces lanuginosus 195 by solid state fermentation (SSF) was purified 9.3-fold from a crude koji extract, with a 7.6% final yield. The purified xylanase (with an estimated mass of 22 kDa by SDS-PAGE) retained 18% relative activity when treated for 10 min at 100 °C and approximately 90% relative activity when incubated at pH values ranging from 6 to 10. Xylanase activity in the purified preparation was significantly enhanced following treatment with manganese and potassium chlorides (p < 0.05) but significantly reduced by calcium, cobalt and iron (p < 0.05). The purified enzyme was also shown to be exclusively xylanolytic. The gene encoding xylanase activity from T. lanuginosus 195 was functionally expressed by Pichia pastoris. MALDI-ToF mass spectrometry and zymography were employed to confirm functional recombinant expression. Maximum xylanase titres were achieved following 120 h induction of the recombinant culture, yielding 26.8 U/mL. Achieving functional protein expression facilitates future efforts to optimise the cultivation conditions for heterologous xylanase production.     

Optimal conditions for enhanced β-mannanase production by recombinant Aspergillus sojae

Optimization of the growth conditions for maximum β-mannanase production in shake flasks by using recombinant Aspergillus sojae ATCC11906 (AsT1) was carried out by Box–Behnken design of response surface methodology. The highest β-mannanase activity on the fourth day of cultivation at 30 °C was obtained as 363 U/ml in the optimized medium consisting of 7% sugar beet molasses, 0.43% NH₄NO₃, 0.1% K₂HPO₄ and 0.05% MgSO₄ (by weight per volume) at 207 rpm. On the sixth day of cultivation under the optimized conditions, the highest β-mannanase activity was achieved as 482 U/ml which is 1.4-fold of 352 U/ml activity found on glucose medium previously.     

Heterologous production of a temperature and pH-stable laccase from Bacillus vallismortis fmb-103 in Escherichia coli and its application

To enhance laccase yield, the laccase gene from Bacillus vallismortis fmb-103 was cloned and heterologously expressed in Escherichia coli BL21 (DE3) cells. The auto-induction strategy was applied during fermentation, and the process was controlled, as follows: Cu²⁺ was added when the optical density at 600 nm (OD₆₀₀) was 0.3, the fermentation temperature was adjusted to 16 °C when the OD₆₀₀ was 0.9, and fermentation was stopped after 50 h. The yield of recombinant laccase was up to 3420 U/L, as assayed by 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Recombinant laccase was purified 4.47-fold by heating for 10 min at 70 °C and dialyzing against 50–60% ammonium sulfate, retained more than 50% activity after 10 h at 70 °C, and demonstrated broad pH stability. Malachite green was efficiently degraded by recombinant laccase, especially in combination with mediators. These results provided a basis for the future application of recombinant laccase to malachite green degradation.     

Expression of cellobiose dehydrogenase from Neurospora crassa in Pichia pastoris and its purification and characterization

A gene encoding cellobiose dehydrogenase (CDH) from Neurospora crassa strain FGSC 2489 has been cloned and expressed in the heterologous host Pichia pastoris, under the control of the AOX1 methanol inducible promoter. Recombinant CDH without the native signal sequence and fused with a His₆-tag (rNC-CDH1) was successfully expressed and secreted. rNC-CDH1 was produced at the level of 652 IU/L after 2 days of cultivation in the induction medium. The His₆-tagged rNC-CDH1 was purified through a one-step Ni–NTA affinity column under non-denaturing conditions. The purified rNC-CDH1 has a CDH activity of 7451 IU/L (0.89 mg protein/mL), with a specific CDH activity of 8.37 IU/mg. The purity of the enzyme was examined by SDS–PAGE, and a single band corresponding to a molecular weight of about 120 kDa was observed. Activity staining confirmed the CDH activity of the protein band. The purified rNC-CDH1 has maximum CDH activity at pH 4.5, and a rather broad temperature optimum of 25–70 °C. Kinetic analysis showed cellobiose and cellooligosaccharides are the best substrates for rNC-CDH1. The K_m value of the rNC-CDH1 for cellooligosaccharide increases with the elongation of glucosyl units. k_cat remains relatively constant when the chain length changes.     

Proteases supplementation to high gravity worts enhances fermentation performance of brewer's yeast

Nitrogen limitation, particularly prevailing in the case of high gravity beer brewing, results in poor yeast viability and even stuck or sluggish fermentations. Although wort contains abundant proteins and longer chain peptides, brewer's yeast does not assimilate them due to the fact that cells hardly secrete proteases during fermentation. The objective of this study was to investigate the possibility for utilizing unavailable nitrogen from two types of high gravity worts (20 °P and 24 °P) by adding three food-grade commercial proteases (Neutrase, Flavorzyme and Protamex) at the beginning of fermentations, respectively. Results showed that proteases supplementation significantly increased the FAN level and thus the amount of cell suspension in the later stages of fermentations (ca. 10 days later for 20 °P and 25 days later for 24 °P) (p < 0.05). Among the studied three proteases, we found that fermentations with Flavorzyme supplementation exhibited the best fermentation performance in terms of significantly improved wort fermentability, higher ethanol yield and flavor volatiles formation (p < 0.05). Furthermore, the foam of final beers produced by adding proteases was as stable as that of the control at each of the corresponding gravities.     

Heterologous expression of codon optimized Trichoderma reesei Cel6A in Pichia pastoris

The Cel6A deficiency has become one of the limiting factors for cellulose saccharification in biochemical conversion of cellulosic biomass to fuels and chemicals. The work attempted to use codon optimization to enhance Trichoderma reesei Cel6A expression in Pichia pastoris. Two recombinants P. pastoris GS115 containing AOX1 and GAP promotors were successfully constructed, respectively. The optimal temperatures and pHs of the expressed Cel6A from two recombinants were consistent with each other, were also in the extremely similar range to that reported on the native Cel6A from T. reesei. Based on the shake flask fermentation, AOX1 promotor enabled the recombinant to produce 265 U/L and 300 mg/L of the Cel6A enzyme, and the GAP promotor resulted in 145 U/L and 200 mg/L. High cell density fed batch (HCDFB) fermentation significantly improved the enzyme titer (1100 U/L) and protein yield (2.0 g/L) for the recombinant with AOX1 promotor. Results have showed that the AOX1 promotor is more suitable than the GAP for the Cel6A expression in P. pastoris. And the HCDFB cultivation is a favorable way to express the Cel6A highly in the methanol inducible yeast.     

Chaperone-dependent gene expression of organic solvent-tolerant lipase from Pseudomonas aeruginosa strain S5

The gene coding for the intracellular organic solvent-tolerant lipase of Pseudomonas aeruginosa strain S5 was isolated from a genomic DNA library and cloned into pRSET. The cloned sequence included two open reading frames (ORF) of 1575 bp for the first ORF (ORF1), and 582 bp for the second ORF (ORF2). The ORF2, known as chaperone, plays an important role in the expression of the S5 gene. The ORF2 is located downstream of lipase gene, and functions as the act gene for ORF1. The conserved pentapeptide, Gly-X-Ser-X-Gly, is located in the ORF1. A sequence coding for a catalytic triad that resembles that of a serine protease, consisting of serine, histidine, and aspartic acid or glutamic acid residues, was present in the lipase gene. Expression of the S5 lipase gene in E. coli resulted in a 100-fold increase in enzyme activity 9 h after induction with 0.75 mM IPTG. The recombinant protein revealed a size of 60 kDa on SDS-PAGE. The Lip S5 gene was stable in the presence of 25% (v/v) n-dodecane and n-tetradecane after 2 h incubation at 37 °C.     

Kinetic properties of glycerophosphate oxidase isolated from dry baker's yeast

The glycerophosphate oxidase is a flavoprotein responsible for the catalysis of the oxidation of the glycerophosphate to dihydroxyacetone phosphate, through the reduction of the oxygen to hydrogen peroxide. The glycerophosphate oxidase from baker's yeast was specific for l-α-glycerol phosphate. It was estimated by monitoring the consumption of oxygen with an oxygraph. An increase of 32% in consumption of oxygen was obtained when the enzyme was concentrated 16-fold. The assay of enzyme was determined by the peroxidase chromogen method followed at 500 nm. The procedure for the standardization of the activity of the glycerophosphate oxidase from baker's yeast was accomplished, and the pH and temperature stability showed that the enzyme presented a high stability at pH 8.0, and the thermal stability was maintained up to 60 °C during 1 h. Such method allowed quantifying in the range 92–230 mM of glycerol phosphate, an important intermediate metabolite from lipid biosynthesis and glycolytic routes.     

Functional characterization of the sucrose isomerase responsible for trehalulose production in plant-associated Pectobacterium species

Fifty-three plant-associated microorganisms were investigated for their ability to convert sucrose to its isomers. These microorganisms included one Dickeya zeae isolate and 7 Enterobacter, 3 Pantoea, and 43 Pectobacterium species. Eleven out of the 53 strains (21%) showed the ability to transform sucrose to isomaltulose and trehalulose. Among those, Pectobacterium carotovorum KKH 3-1 showed the highest bioconversion yield (97.4%) from sucrose to its isomers. In this strain, the addition of up to 14% sucrose in the medium enhanced sucrose isomerase (SIase) production. The SIase activity at 14% sucrose (47.6 U/mg dcw) was about 3.6-fold higher than that of the negative control (13.3 U/mg dcw at 0% sucrose). The gene encoding SIase, which is comprised a 1776 bp open reading frame (ORF) encoding 591 amino acids, was cloned from P. carotovorum KKH 3-1 and expressed in Escherichia coli. The recombinant SIase (PCSI) was shown to have optimum activity at pH 6.0 and 40 °C. The reaction temperature significantly affected the ratio of sucrose isomers produced by PCSI. The amount of trehalulose increased from 47.5% to 79.1% as temperature was lowered from 50 °C to 30 °C, implying that SIase activity can be controlled by reaction temperature.     

The effect of acclimation temperature on thermal activity thresholds in polar terrestrial invertebrates

In the Maritime Antarctic and High Arctic, soil microhabitat temperatures throughout the year typically range between ?10 and +5 °C. However, on occasion, they can exceed 20 °C, and these instances are likely to increase and intensify as a result of climate warming. Remaining active under both cool and warm conditions is therefore important for polar terrestrial invertebrates if they are to forage, reproduce and maximise their fitness. In the current study, lower and upper thermal activity thresholds were investigated in the polar Collembola, Megaphorura arctica and Cryptopygus antarcticus, and the mite, Alaskozetes antarcticus. Specifically, the effect of acclimation on these traits was explored. Sub-zero activity was exhibited in all three species, at temperatures as low as ?4.6 °C in A. antarcticus. At high temperatures, all three species had capacity for activity above 30 °C and were most active at 25 °C. This indicates a comparable spread of temperatures across which activity can occur to that seen in temperate and tropical species, but with the activity window shifted towards lower temperatures. In all three species following one month acclimation at ?2 °C, chill coma (=the temperature at which movement and activity cease) and the critical thermal minimum (=low temperature at which coordination is no longer shown) occurred at lower temperatures than for individuals maintained at +4 °C (except for the CTmin of M. arctica). Individuals acclimated at +9 °C conversely showed little change in their chill coma or CTmin. A similar trend was demonstrated for the heat coma and critical thermal maximum (CTmax) of all species. Following one month at ?2 °C, the heat coma and CTmax were reduced as compared with +4 °C reared individuals, whereas the heat coma and CTmax of individuals acclimated at +9 °C showed little adjustment. The data obtained suggest these invertebrates are able to take maximum advantage of the short growing season and have some capacity, in spite of limited plasticity at high temperatures, to cope with climate change.     

Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase

Improvement of thermostability in engineered enzymes can allow biocatalysis on substrates with poor aqueous solubility. Denaturation of the cofactor-binding loops of Escherichia coli transketolase (TK) was previously linked to the loss of enzyme activity under conditions of high pH or urea. Incubation at temperatures just below the thermal melting transition, above which the protein aggregates, was also found to anneal the enzyme to give an increased specific activity. The potential role of cofactor-binding loop instability in this process remained unclear. In this work, the two cofactor-binding loops (residues 185–192 and 382–392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity. Cofactor-binding loop 2 variants had detrimental effects on specific activity at elevated temperatures, whereas the H192P mutation in cofactor-binding loop 1 resulted in a two-fold improved stability to inactivation at elevated temperatures, and increased the critical onset temperature for aggregation. The specific activity of H192P was 3-fold and 19-fold higher than that for wild-type at 60 °C and 65 °C respectively, and also remained 2.7-4 fold higher after re-cooling from pre-incubations at either 55 °C or 60 °C for 1 h. Interestingly, H192P was also 2-times more active than wild-type TK at 25 °C. Optimal activity was achieved at 60 °C for H192P compared to 55 °C for wild type. These results show that cofactor-binding loop 1, plays a pivotal role in partial denaturation and aggregation at elevated temperatures. Furthermore, a single rigidifying mutation within this loop can significantly improve the enzyme specific activity, as well as the stability to thermal denaturation and aggregation, to give an increased temperature optimum for activity.     

Heat shock at higher cell densities improves measles hemagglutinin translocation and human GRP78/BiP secretion in Saccharomyces cerevisiae

The yield of heterologous proteins is often limited by several bottlenecks in the secretory pathway of yeast Saccharomyces cerevisiae. It was shown earlier that synthesis of measles virus hemagglutinin (MeH) is inefficient mostly due to a bottleneck in the translocation of viral protein precursors into the endoplasmic reticulum (ER) of yeast cells. Here we report that heat shock with subsequent induction of MeH expression at 37 °C improved translocation of MeH precursors when applied at higher cell densities. The amount of MeH glycoprotein increased by about 3-fold after heat shock in the late-log phases of both glucose and ethanol growth. The same temperature conditions increased both secretion titer and yield of another heterologous protein human GRP78/BiP by about 50%. Furthermore, heat shock at the late-log glucose growth phase also improved endogenous invertase yield by approximately 2.7-fold. In contrast, a transfer of yeast culture to lower temperature at diauxic shift followed by protein expression at 20 °C almost totally inhibited translocation of MeH precursors. The difference in amounts of MeH glycoprotein under expression at 37 °C and 20 °C was about 80-fold, while amounts of unglycosylated MeH polypeptides were similar under both conditions. Comparative proteomic analysis revealed that besides over-expressed ER-resident chaperone Kar2, an increased expression of several cytosolic proteins (such as Hsp104, Hsp90 and eEF1A) may contribute to improved translocation of MeH.     

Cloning,expression, purification and characterization of an oligomeric His-tagged thermophilic esterase from Thermus thermophilus HB27

The esterase E34Tt (YP_004875.1) from Thermus thermophilus HB27 was cloned, expressed in Escherichia coli as a His-tagged protein, purified and characterized. The gene sequence was subcloned into a T-vector, released with the restriction enzymes BamHI and HindIII, ligated to a pET-21d(+) vector, and transferred to E. coli BL21 (DE3) cells. Inducer concentration (isopropyl β-d-1-thiogalactopyranoside, IPTG) and cultivation time before and after induction were optimized. Best results were obtained by adding 0.25 mM IPTG after 8 h of cultivation and maintaining the induction during 4 extra hours. Most of the enzyme (94%) remained membrane-associated and had to be extracted with a detergent. From the membrane crude extract, the His-tagged E34Tt was purified as a dimer (71.8 kDa) in a single purification step by using metal affinity chromatography. The Rosso's model was used to optimize the reaction conditions. E34Tt-His₆ was active in a wide temperature (19.7–79.4 °C) and pH range (4.0–9.3), and maximal activity was determined at pH 6.3 and 58.2 °C, which is 10–18 °C higher than the optimal reaction temperature of the previously reported variants expressed in mesophilic yeasts. E34Tt-His₆ preferentially hydrolyzed esters with ten carbon atoms, and was highly thermostable (half-life of 107.9 min at 85 °C), suggesting that E34Tt-His₆ has potential for industrial applications.     

Mathematical modeling of Microcystis aeruginosa growth and [D-Leu1] microcystin-LR production in culture media at different temperatures

The effect of temperature (26 °C, 28 °C, 30 °C and 35 °C) on the growth of native CAAT-3-2005 Microcystis aeruginosa and the production of Chlorophyll-a (Chl-a) and Microcystin-LR (MC-LR) were examined through laboratory studies. Kinetic parameters such as specific growth rate (μ), lag phase duration (LPD) and maximum population density (MPD) were determined by fitting the modified Gompertz equation to the M. aeruginosa strain cell count (cells mL⁻¹). A 4.8-fold increase in μ values and a 10.8-fold decrease in the LPD values were found for M. aeruginosa growth when the temperature changed from 15 °C to 35 °C. The activation energy of the specific growth rate (Eμ) and of the adaptation rate (E₁/LPD) were significantly correlated (R² = 0.86). The cardinal temperatures estimated by the modified Ratkowsky model were minimum temperature = 8.58 ± 2.34 °C, maximum temperature = 45.04 ± 1.35 °C and optimum temperature = 33.39 ± 0.55 °C.Maximum MC-LR production decreased 9.5-fold when the temperature was increased from 26 °C to 35 °C. The maximum production values were obtained at 26° C and the maximum depletion rate of intracellular MC-LR was observed at 30–35 °C. The MC-LR cell quota was higher at 26 and 28 °C (83 and 80 fg cell⁻¹, respectively) and the MC-LR Chl-a quota was similar at all the different temperatures (0.5–1.5 fg ng⁻¹).The Gompertz equation and dynamic model were found to be the most appropriate approaches to calculate M. aeruginosa growth and production of MC-LR, respectively. Given that toxin production decreased with increasing temperatures but growth increased, this study demonstrates that growth and toxin production processes are uncoupled in M. aeruginosa. These data and models may be useful to predict M. aeruginosa bloom formation in the environment.     

Elicitation with abiotic stresses improves pro-health constituents,antioxidant potential and nutritional quality of lentil sprouts

Phenolic content and antioxidant potential of lentil sprouts may be enhanced by treatment of seedlings in abiotic stress conditions without any negative influence on nutritional quality.The health-relevant and nutritional quality of sprouts was improved by elicitation of 2-day-old sprouts with oxidative, osmotic, ion-osmotic and temperature stresses. Among the sprouts studied, those obtained by elicitation with osmotic (600 mM mannitol) and ion-osmotic (300 mM NaCl) shocks had the highest total phenolic content levels: 6.52 and 6.56 mg/g flour, respectively. Oxidative stress significantly enhanced the levels of (+)-catechin and p-coumaric acid. A marked elevation of the chlorogenic and gallic acid contents was also determined for sprouts induced at 4 °C and 40 °C. The elevated phenolic content was translated into the antioxidant potential of sprouts, especially the ability to reduce lipid oxidation. A marked elevation of this ability was determined for seedlings treated with 20 mM, 200 mM H₂O₂ (oxidative stress) and 600 mM mannitol (osmotic stress); about a 12-fold, 8-fold and 9.5-fold increase in respect to control sprouts. The highest ability to quench free radicals was observed in sprouts induced by osmotic stress (IC₅₀- 4.91 and 5.12 mg/ml for 200 mM and 600 mM mannitol, respectively). The highest total antioxidant activity indexes were determined for sprouts elicited with 20 mM H₂O₂ and 600 mM mannitol: 4.0 and 3.4, respectively. All studied growth conditions, except induction at 40 °C, caused a significant elevation of resistant starch levels which was also affected in a subsequent reduction of starch digestibility.Improvement of sprout quality by elicitation with abiotic stresses is a cheap and easy biotechnology and it seems to be an alternative to conventional techniques applied to improve the health promoting phytochemical levels and bioactivity of low-processed food.